A partially purified plant protease preparation is 40% inactivated by treatment

Question

A partially purified plant protease preparation is 40% inactivated by treatment with iodoacetate (IAA) and 100% inactivated by treatment with N-tosyl-L-lysine chloromethylketone (TLCK). When 14C-labeled TLCK is employed the loss of activity is accompanied by the incorporation of counts into two polypeptides, one that migrates at Mr 50,000 and another that migrates at Mr 35,000 upon SDS-PAGE.

N-tosyl-L-lysine chloromethylketone (TLCK)

Iodoacetate (IAA)

A. On the basis of these findings, which residues likely directly participate in the proteolytic reaction(s) catalyzed by this protease preparation? What assumptions must you make concerning the primary and secondary effects of these reagents in order to draw this(these) conclusion(s)? Which residues or functional groups is each reagent specific for and and in what context?

B. How would you further test your proposal(s)? Again, explain your assumptions and any limitations inherent in the techniques you suggest. It will be particularly important to design an experiment or experiments that enable you to gain an indication of whether the residues you have invoked in the catalytic mechanism are dependent on each other or capable of acting independently (for instance, whether two different residues on the same or different polypeptides might participate in the same or different proteolytic reactions). It is intriguing that treatment of the protease preparation with [14C]TLCK labels two polypeptides.

C. If you were to discover that diisopropylphosphofluoridate (DIPF) abolishes 60% of the total proteolytic activity of the preparation and that the DIPF-inhibitable component is also subject to inhibition by TLCK, what would you be forced to conclude? How would you further test your conclusion(s)? Assume that you also have a ready source of [32P]DIPF.

Explanation / Answer

Proteases are the main enzymes controling cellular processes, they are present all over in all cells as well as tissues.Proteases are liberated into the lysate upon cell lysis . Some of the proteases create a significant restraint to the biochemical analysis. They can produce false results treating the proteins location, activity or structure. Protease inhibitors are applied in order to stop degradation of involuntary protein. Protease inhibitors act on their target proteins either reversibly or irreversibly. Reversible binding of inhibitors may be extinct during dialysis or various purification steps.  N-tosyl-L-lysine chloromethylketone (TLCK) hinder trypsin irreversibly by alkylation of histidine residue in the enzyme active site. Iodoacetate is also an irreversible cysteine peptidases inhibitor, with the inhibition process generating from alkylation of the cysteine residue. Therefore,100% inactivation by treatment with (TLCK) shows that purified protease contains more trypsin protease that are used to hydrolyse the peptide bonds of the basic amino acids ( lysine, arginine) at the C terminal.

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